Methods to detect a silent carrier of a null allele genotype

ABSTRACT

Provided herein are methods and compositions for the detection of silent carriers of chromosomal deletion alleles in a human subject using haploid cells (e.g., sperm cells or egg cells) derived from the subject. The methods provided herein comprise quantitative nucleic acid amplification reactions and determination of a ratio of a target gene to a reference gene, and indicate that a subject is a silent carrier of a null allele corresponding to the target gene if the ratio is at or below a threshold level. The methods provided herein allow for the detection of silent (2+0) carriers of SMA, where the individual has a deletion of the SMN1 gene on one chromosome 5 homolog and two or more copies of the SMN1 gene on other chromosome 5 homolog.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 62/080,047 filed Nov. 14, 2014, the contents of whichare incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 31, 2015, isnamed 103779-0672 SL.txt and is 1,984 bytes in size.

BACKGROUND OF THE INVENTION

Spinal muscular atrophy (SMA) is the second most common fatal autosomalrecessive disorder after cystic fibrosis, affecting approximately 1 in6,000 to 10,000 live births. The disorder is characterized by hypotonia,proximal muscle weakness and respiratory distress due to degeneration ofmotor neurons in the spinal cord. SMA is caused by mutations in thesurvival motor neuron 1 (SMN1) gene, which is located on chromosome 5 at5q11.2-13.3. The majority of affected individuals exhibit loss of theSMN1 gene, either by complete gene deletion or through a gene conversionevent involving the adjacent SMN2 gene. The SMN2 gene differs from theSMN1 gene by a single nucleotide (840C>T) in exon 7 and lies in aninverted orientation in cis- with the SMN1 gene on chromosome 5. Atleast one copy of the SMN1 gene is indispensable for normal survival ofmotor neurons. In contrast, both copies of the SMN2 gene are dispensableas approximately 5-10% of normal individuals lack both copies of SMN2,though in some cases, the number of SMN2 copies can modulate theclinical phenotype.

The molecular diagnosis of SMA is generally accomplished through thedetection of a homozygous deletion of SMN1. More than 95% of SMA patentshave a homozygous deletion of SMN1 exon 7. Carrier testing for SMA,however, is particularly challenging for several reasons. Because theSMN1 gene is highly homologous to SMN2, abnormalities in the SMN1 genecan only be detected with carefully designed allele-specific assays.Further, in about 4% of the carrier population, a chromosomal alterationplaces both copies of the SMN1 gene on one chromosome and zero copies onthe other (i.e., silent carrier or 2+0 genotype). Gene dosage analysiscan determine the copy number of SMN1 to detect carrier status inindividuals that are heterozygous for the absence of SMN1, but areineffective for detecting silent carrier genotypes, where two copies ofthe SMN1 gene are present on only one chromosome. In addition, becausethe SMN1 and SMN2 genes are separated by a long distance (800 kb) on thesame chromosome, linkage analysis of the chromosomal defect isdifficult.

SUMMARY OF THE INVENTION

Described herein, in certain embodiments, are methods and compositionsfor the detection of silent carriers of chromosomal deletion alleles ina human subject using haploid cells derived from the subject. In someembodiments, the haploid cells are gametes (e.g., sperm cells or eggcells). In particular embodiments, the methods provided herein allow forthe detection of silent (2+0) carriers of SMA, where the individual hasa deletion of the SMN1 gene on one chromosome 5 homolog and two or morecopies of the SMN1 gene on other chromosome 5 homolog.

Provided herein, in certain embodiments, are methods for identifying asubject as a silent carrier of a target gene null allele. In someembodiments, the method involves (a) performing a plurality of nucleicacid amplification reactions, wherein each nucleic acid amplificationreaction comprises a genomic DNA sample obtained from a single haploidcell from the subject, at least one pair of oligonucleotide primers foramplification of a target region of a target gene for the generation ofa target gene amplification product, wherein the region amplified in thetarget gene amplification product is deleted in the target gene nullallele, and at least one pair of oligonucleotide primers foramplification of a target region of a reference gene for the generationof a reference gene amplification product; (b) detecting the presence orabsence of the target gene amplification product; (c) detecting thepresence or absence of the reference gene amplification product; and (d)characterizing the subject as a carrier of the target gene null alleleif the ratio of target gene amplification products to reference geneamplification products is at or below a threshold level. In someembodiments, the threshold level is between about 0.5 and about 0.8. Forexample, in some embodiments, the threshold level is threshold level isabout 0.75 or about 0.8. In some embodiments, the ratio of target geneamplification products to reference gene amplification products in asilent carrier of a target gene null allele is approximately 0.5. Themethods provided herein are typically performed on sample obtained froma mammalian subject, and particularly a human subject. In particularembodiments, the target gene for amplification is SMN1. In someembodiments, the target gene amplification product contains exon 7 ofSMN1 or a portion thereof. In some embodiments, the reference gene isselected from among CFTR, GAPDH, HMBS, B2M, HPRT1, RPL13A, SDHA, TBP,UBC, YWHAZ, PRDX6, ADD1, HLA-A, RAD9A, ARHGEF7, EIF2B2, PSMD7, BCAT2,and ATP5O. In particular embodiments, the reference gene is CFTR. Insome embodiments, homozygous deletion of the target gene is associatedwith a disease or condition. In some embodiments, disease or conditionis spinal muscular atrophy (SMA).

Exemplary haploid cells for use in the methods include a naturallyoccurring gamete cells or induced haploid cells. In some embodiments,the haploid cell is a sperm cell or an egg cell. In some embodiments,where the haploid cell is an induced haploid cell, the haploid cell isderived from an induced pluripotent stem cell (iPSC). In someembodiments, the iPSC is generated from an adult stem cell from thesubject.

In some embodiments, at least one oligonucleotide primer of the primerpair for amplification of the target region of the target gene and/orthe reference gene is labeled with a detectable moiety, such as such asa radioactive moiety, a fluorescent moiety, or a dye molecule. In someembodiments, the nucleic acid amplification reaction is polymerase chainreaction (PCR) or particularly quantitative PCR. In some embodiments,each nucleic acid amplification reaction is performed in a separate wellof a multiwell plate. In some embodiments, the target gene amplificationproduct and/or the reference gene amplification product is detected witha labeled nucleic acid probe specific for the target gene amplificationproduct.

In some embodiments, the methods provided involve a step of preparingthe genomic DNA from single haploid cells. In an exemplary method,preparing the genomic DNA from single haploid cells involves: (a)sorting single haploid cells into separate reaction vessels at aconcentration of one haploid cell per reaction vessel; and (b)contacting each sorted cell with a lysis buffer to release the genomicDNA from the cell. In some embodiments, the lysis buffer comprises anenzyme to assist in lysis of the haploid cell. For example, in someembodiments, the lysis buffer comprises a protease. In some embodiments,the lysis buffer comprises proteinase K. Preparation of the genomic DNAand the nucleic acid amplification reaction can be performed in the samereaction vessel or separate reaction vessels. Preparation of the genomicDNA and the nucleic acid amplification reaction in the same reactionvessel minimizes loss of genomic DNA. In some embodiments, the reactionvessel is a well of a microtiter plate, a microchip or reaction gridslide.

In some embodiments, the methods involve droplet digital PCR. In someembodiments, each haploid cell to be analyzed is first encapsulated inan microdroplet. In some embodiments, the microdroplets are dispersed inan aqueous-in-oil emulsion in a single vessel. In some embodiments, themicrodroplets are sorted into individual vessels. In some embodiments,each haploid cell is lysed within the microdroplet. In some embodiments,the microdroplets containing the lysed cells are then subjected to anucleic acid amplification reaction. In some embodiments, the nucleicacid amplification products are detected within the microdroplets. Inother embodiments, the amplification products are isolated from themicrodroplets and detected.

In some embodiments, the methods further involve determining the copynumber of the target gene in a diploid cell from the test subject. Insome embodiments, the methods further involve generating cell line fromdiploid cells of the test subject. In some embodiments, the methodsfurther involve sequencing the SMN1 and/or SMN2 gene or portions thereof

In some embodiments, the methods further involve generation of a report,wherein the report contains an assessment of the likelihood that thesubject is a silent carrier of the target gene null allele.

Also provided herein are kits for the performing the methods describedherein. In an exemplary embodiment a kit for the performance of themethods provided contains: (a) a pair of oligonucleotide primersspecific to the SMN1 gene for the generation of a target geneamplification product, wherein the region amplified in the SMN1 geneamplification product is deleted in an SMN1 silent carrier, and (b) apair of oligonucleotide primers specific to a reference gene for thegeneration of a reference gene amplification product that is not deletedin an SMN1 silent carrier; and (c) one or more reagents for performing anucleic acid amplification reaction. In some embodiments, the kitcomprises nucleotide triphosphates, a thermostable polymerase, and/or asuitable buffer. In some embodiments, the reference gene is selectedfrom among CFTR, GAPDH, HMBS, B2M, HPRT1, RPL13A, SDHA, TBP, UBC, YWHAZ,PRDX6, ADD1, HLA-A, RAD9A, ARHGEF7, EIF2B2, PSMD7, BCAT2, and ATP5O. Insome embodiments, the target gene amplification product comprises exon 7of SMN1 or a portion thereof.

Also provided herein are microtiter plates for the performing themethods described herein. Exemplary microtiter plates contain aplurality of reaction vessels (e.g., wells), wherein one or morereaction vessels of the microtiter plate contain: (a) a pair ofoligonucleotide primers specific to the SMN1 gene for the generation ofa target gene amplification product, wherein the region amplified in theSMN1 gene amplification product is deleted in an SMN1 silent carrier;(b) a pair of oligonucleotide primers specific to a reference gene forthe generation of a reference gene amplification product that is notdeleted in an SMN1 silent carrier; and (c) one or more reagents forperforming a nucleic acid amplification reaction. In some embodiments,the one or more reaction vessels comprises nucleotide triphosphates, athermostable polymerase, and/or a suitable buffer. In some embodiments,the reference gene is selected from among CFTR, GAPDH, HMBS, B2M, HPRT1,RPL13A, SDHA, TBP, UBC, YWHAZ, PRDX6, ADD1, HLA-A, RAD9A, ARHGEF7,EIF2B2, PSMD7, BCAT2, and ATP5O. In some embodiments, the target geneamplification product comprises exon 7 of SMN1 or a portion thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the organization of SMN1 and SMN2 gene loci onchromosome 5 at 5q13. Provided are details of the chromosomalarrangement of the SMN1 and SMN2 gene copies relative to normal,carrier, and silent carrier genotypes.

FIG. 2 illustrates an exemplary assay workflow of the single sperm cellqPCR assay.

FIG. 3 illustrates data for detecting the SMN1 gene in a singlespermatozoa qPCR Assay. The average value of single cell qPCR assayratio values of SMN1 versus a reference gene and both gene targetsversus a reference gene are shown. *P<0.01. Standard deviation ofobserved values is indicated by error bars.

FIG. 4 illustrates resolution of an identified 2+0 Genotype Result. (A)Average value of single cell qPCR assay ratio values of SMN1 versus areference gene and both gene targets versus a reference gene forspecimen DS11. (B) Non-specific sequencing of the SMN1 and SMN2 genes,+6 position of exon 7 c.840C>T highlighted by red box (SEQ ID NOS 6 and6, respectively, in order of appearance). (C) Specific sequencing ofSMN1 qPCR primer and probe sites (SEQ ID NOS 7 and 7, respectively, inorder of appearance). *P<0.001. Standard deviation of observed valuedindicated by error bars.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Certain Terminology

To facilitate an understanding of the present disclosure, a number ofterms and phrases are defined below.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” also include the plural. Thus, for example, a reference to “anoligonucleotide” includes a plurality of oligonucleotide molecules, areference to “a label” is a reference to one or more labels, a referenceto “a probe” is a reference to one or more probes, and a reference to “anucleic acid” is a reference to one or more polynucleotides.

As used herein, unless indicated otherwise, when referring to anumerical value, the term “about” means plus or minus 10% of theenumerated value.

As used herein, a “carrier” or “genetic carrier” is an individual havingat least one copy of an allele of a genetic determinant that is involvedin the expression of a particular phenotype, such as SMA.

As used herein, a “silent carrier” is a genetic carrier that cannot bedetected using a copy number-based diagnostic technique. For example, a“silent carrier” is a genetic carrier that has a deletion of all or partof a target gene on one chromosome homolog and two or more copies of thetarget gene on the other chromosome homolog.

As used herein an “SMA silent carrier” or an “SMA (2+0) carrier is agenetic carrier that has a deletion of all or part of the SMN1 gene onone chromosome 5 homolog and two or more copies of the SMN1 gene on theother chromosome 5 homolog.

The terms “amplification” or “amplify” as used herein includes methodsfor copying a target nucleic acid, thereby increasing the number ofcopies of a selected nucleic acid sequence. Amplification may beexponential or linear. A target nucleic acid may be either DNA or RNA.The sequences amplified in this manner form an “amplification product,”also known as an “amplicon.” While the exemplary methods describedhereinafter relate to amplification using the polymerase chain reaction(PCR), numerous other methods are known in the art for amplification ofnucleic acids (e.g., isothermal methods, rolling circle methods, etc.).The skilled artisan will understand that these other methods may be usedeither in place of, or together with, PCR methods. See, e.g., Saiki,“Amplification of Genomic DNA” in PCR Protocols, Innis et al., Eds.,Academic Press, San Diego, Calif. 1990, pp. 13-20; Wharam et al.,Nucleic Acids Res., 29(11):E54-E54, 2001; Hafner et al., Biotechniques,30(4):852-56, 858, 860, 2001; Zhong et al., Biotechniques, 30(4):852-6,858, 860, 2001.

As used herein, the term “detecting” refers to observing a signal from adetectable label to indicate the presence of a target. Morespecifically, detecting is used in the context of detecting a specificsequence.

The terms “complement,” “complementary” or “complementarity” as usedherein with reference to polynucleotides (i.e., a sequence ofnucleotides such as an oligonucleotide or a genomic nucleic acid)related by the base-pairing rules. The complement of a nucleic acidsequence as used herein refers to an oligonucleotide which, when alignedwith the nucleic acid sequence such that the 5′ end of one sequence ispaired with the 3′ end of the other, is in “antiparallel association.”For example, for the sequence 5′-A-G-T-3′ is complementary to thesequence 3′-T-C-A-5′. Certain bases not commonly found in naturalnucleic acids may be included in the nucleic acids of the presentdisclosure and include, for example, inosine and 7-deazaguanine.Complementarity need not be perfect; stable duplexes may containmismatched base pairs or unmatched bases. Those skilled in the art ofnucleic acid technology can determine duplex stability empiricallyconsidering a number of variables including, for example, the length ofthe oligonucleotide, base composition and sequence of theoligonucleotide, ionic strength and incidence of mismatched base pairs.Complementarity may be “partial” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete,” “total,” or “full” complementarity between thenucleic acids.

The term “detectable label” as used herein refers to a molecule or acompound or a group of molecules or a group of compounds associated witha probe and is used to identify the probe hybridized to a genomicnucleic acid or reference nucleic acid.

A “fragment” in the context of a polynucleotide refers to a sequence ofnucleotide residues, either double- or single-stranded, which are atleast about 2 nucleotides, at least about 5 nucleotides, at least about10 nucleotides, at least about 20 nucleotides, at least about 25nucleotides, at least about 30 nucleotides, at least about 40nucleotides, at least about 50 nucleotides, at least about 100nucleotides.

The terms “identity” and “identical” refer to a degree of identitybetween sequences. There may be partial identity or complete identity. Apartially identical sequence is one that is less than 100% identical toanother sequence. Partially identical sequences may have an overallidentity of at least 70% or at least 75%, at least 80% or at least 85%,or at least 90% or at least 95%.

As used herein, the terms “isolated,” “purified” or “substantiallypurified” refer to molecules, such as nucleic acid, that are removedfrom their natural environment, isolated or separated, and are at least60% free, preferably 75% free, and most preferably 90% free from othercomponents with which they are naturally associated. An isolatedmolecule is therefore a substantially purified molecule.

As used herein, the term “oligonucleotide” or “polynucleotide” refers toa short polymer composed of deoxyribonucleotides, ribonucleotides, orany combination thereof. Oligonucleotides are generally between about10, 11, 12, 13, 14, 15, 20, 25, or 30 to about 150 nucleotides (nt) inlength, more preferably about 10, 11, 12, 13, 14, 15, 20, 25, or 30 toabout 70 nt.

As used herein, a “primer” is an oligonucleotide that is complementaryto a target nucleotide sequence and leads to addition of nucleotides tothe 3′ end of the primer in the presence of a DNA or RNA polymerase. The3′ nucleotide of the primer should generally be identical to the targetsequence at a corresponding nucleotide position for optimal extensionand/or amplification. The term “primer” includes all forms of primersthat may be synthesized including peptide nucleic acid primers, lockednucleic acid primers, phosphorothioate modified primers, labeledprimers, and the like. As used herein, a “forward primer” is a primerthat is complementary to the anti-sense strand of DNA. A “reverseprimer” is complementary to the sense-strand of DNA.

An oligonucleotide (e.g., a probe or a primer) that is specific for atarget nucleic acid will “hybridize” to the target nucleic acid undersuitable conditions. As used herein, “hybridization” or “hybridizing”refers to the process by which an oligonucleotide single strand annealswith a complementary strand through base pairing under definedhybridization conditions. It is a specific, i.e., non-random,interaction between two complementary polynucleotides. Hybridization andthe strength of hybridization (i.e., the strength of the associationbetween the nucleic acids) is influenced by such factors as the degreeof complementary between the nucleic acids, stringency of the conditionsinvolved, and the Tm of the formed hybrid.

“Specific hybridization” is an indication that two nucleic acidsequences share a high degree of complementarity. Specific hybridizationcomplexes form under permissive annealing conditions and remainhybridized after any subsequent washing steps. Permissive conditions forannealing of nucleic acid sequences are routinely determinable by one ofordinary skill in the art and can occur, for example, at 65° C. in thepresence of about 6×SSC. Stringency of hybridization may be expressed,in part, with reference to the temperature under which the wash stepsare carried out. Such temperatures are typically selected to be about 5°C. to 20° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the targetsequence hybridizes to a perfectly matched probe. Equations forcalculating Tm and conditions for nucleic acid hybridization are knownin the art.

As used herein, an oligonucleotide is “specific” for a nucleic acid ifit is capable of hybridizing to the target of interest and notsubstantially hybridizing to nucleic acids which are not of interest.High levels of sequence identity are preferred and include at least 75%,at least 80%, at least 85%, at least 90%, at least 95% and morepreferably at least 98% sequence identity. Sequence identity can bedetermined using a commercially available computer program with adefault setting that employs algorithms well known in the art (e.g.,BLAST).

As used herein, the term “region of interest” or “target region” refersto a region of a nucleic acid to be amplified.

The term “emulsion droplet” or “emulsion microdroplet” refers to adroplet that is formed when two immiscible fluids are combined. Forexample, an aqueous droplet can be formed when an aqueous fluid is mixedwith a non-aqueous fluid. In another example, a non-aqueous fluid can beadded to an aqueous fluid to form a droplet. Droplets can be formed byvarious methods, including methods performed by microfluidics devices orother methods, such as injecting one fluid into another fluid, pushingor pulling liquids through an orifice or opening, forming droplets byshear force, etc. The droplets of an emulsion may have any uniform ornon-uniform distribution. Any of the emulsions disclosed herein may bemonodisperse (composed of droplets of at least generally uniform size),or may be polydisperse (composed of droplets of various sizes). Ifmonodisperse, the droplets of the emulsion may vary in volume by astandard deviation that is less than about plus or minus 100%, 50%, 20%,10%, 5%, 2%, or 1% of the average droplet volume. Droplets generatedfrom an orifice may be monodisperse or polydisperse. An emulsion mayhave any suitable composition. The emulsion may be characterized by thepredominant liquid compound or type of liquid compound that is used. Thepredominant liquid compounds in the emulsion may be water and oil. “Oil”is any liquid compound or mixture of liquid compounds that is immisciblewith water and that has a high content of carbon. In some examples, oilalso may have a high content of hydrogen, fluorine, silicon, oxygen, orany combination thereof, among others. For example, any of the emulsionsdisclosed herein may be a water-in-oil (W/O) emulsion (i.e., aqueousdroplets in a continuous oil phase). The oil may be or include at leastone silicone oil, mineral oil, fluorocarbon oil, vegetable oil, or acombination thereof, among others. Any other suitable components may bepresent in any of the emulsion phases, such as at least one surfactant,reagent, sample (i.e., partitions thereof), buffer, salt, ionic element,other additive, label, particles, or any combination thereof.

As used herein, the term “droplet” refers to a small volume of liquid,typically with a spherical shape or as a slug that fills the diameter ofa microchannel, encapsulated by an immiscible fluid. The volume of adroplet, and/or the average volume of droplets in an emulsion, may beless than about one microliter (i.e., a “microdroplet”) (or betweenabout one microliter and one nanoliter or between about one microliterand one picoliter), less than about one nanoliter (or between about onenanoliter and one picoliter), or less than about one picoliter (orbetween about one picoliter and one femtoliter), among others. A dropletmay have a diameter (or an average diameter) of less than about 1000,100, or 10 micrometers, or of about 1000 to 10 micrometers, amongothers. A droplet may be spherical or nonspherical. In some embodiments,the droplet has a volume and diameter that is large enough toencapsulate a cell. In some embodiments, the droplet has a volume anddiameter that is large enough to encapsulate a haploid cell. In someembodiments, the droplet has a volume and diameter that is large enoughto encapsulate a sperm cell.

The term “bulk sequencing” or “next generation sequencing” or “massivelyparallel sequencing” refers to any high throughput sequencing technologythat parallelizes the DNA sequencing process. For example, bulksequencing methods are typically capable of producing more than onemillion polynucleic acid amplicons in a single assay. The terms “bulksequencing,” “massively parallel sequencing,” and “next generationsequencing” refer only to general methods, not necessarily to theacquisition of greater than 1 million sequence tags in a single run. Anybulk sequencing method can be implemented in the invention, such asreversible terminator chemistry (e.g., Illumina), pyrosequencing usingpolony emulsion droplets (e.g., Roche), ion semiconductor sequencing(IonTorrent), single molecule sequencing (e.g., Pacific Biosciences),massively parallel signature sequencing, etc.

As used herein, the term “subject” refers to a mammal, such as a humanor non-human primate, but can also be another animal such as a domesticanimal (e.g., a dog, cat, or the like), a farm animal (e.g., a cow, asheep, a pig, a horse, or the like) or a laboratory animal (e.g., amonkey, a rat, a mouse, a rabbit, a guinea pig, or the like). The term“patient” refers to a “subject” who possesses, or is suspected topossess, a genetic polymorphism of interest.

Overview of the Silent Carrier Genotyping Assay

Provided herein are methods for genotyping disorders where at least onechromosome of a homologous pair is lacking a gene of interest. Incertain instances, the methods involve determining the carrier statusfor genes that have the propensity for chromosomal rearrangement thatcan leave one chromosome null for the gene. In certain instances, themethods involve detection of the presence or absence of that target geneof interest in a haploid cell, such as a gamete cell (e.g., a sperm oran egg cell). Because gametes are naturally haploid, the existence of achromosome that is null for the target gene can be detected by singlecell genomic analytical methods, such as by nucleic acid amplification.In some embodiments, an induced haploid cell (e.g., by induced germ celldifferentiation of pluripotent stem cells) can also be employed.

Conventional genotyping is performed using diploid cells, most commonlylymphocytes (white blood cells) isolated from whole blood. Such methodsare ineffective for determining the carrier status of the 2+0 silentcarrier genotype because the carrier has two copies of the normal gene.Testing for genetic disorders with a single haploid cell as the sourceremoves the complication of having two copies of all autosome genes aspotential templates for analysis.

In exemplary methods, a single haploid cell is delivered to a reactionvessel and treated (e.g., lysed) to make the genomic DNA of the cellaccessible for the performance of a genetic assay (e.g., PCR). In otherexemplary methods, single haploid cell cells are encapsulated inmicrodroplets (i.e. one cell per droplet), for example, in microdropletsof an aqueous-in-oil emulsion, agarose droplet-in-oil emulsion orembedded in alginate microspheres (see, e.g., Clausell-Tormos et al.(2008) Chem. Biol. 15:427-437). In such embodiments, the cells aretreated (e.g., lysed) within the microdroplets to make the genomic DNAof the cell accessible for the performance of a genetic assay (e.g.,PCR).

In some embodiments, the assay involves nucleic acid amplification ofthe target gene and a reference gene that is present in the cell (e.g.,a house-keeping gene) within the same reaction vessel or microdroplet,and analysis of the amplification products produced. In some embodimentsa plurality of microdroplets, each containing a single cell, iscontained in a single reaction vessel and a nucleic acid amplificationis performed within each microdroplet. The presence or absence of thetarget gene with respect to the reference gene, which should always bepresent, is determined from multiple reactions, each reactionrepresenting the genetic status a single haploid cell. Statistics can beused to analyze the replicate reactions to determine carrier status. Insome embodiments, the amplification products are labeled. In someembodiments, the amplification products are labeled using a primer pairfor amplification in which at least one primer of the primer pair islabeled with a detectable moiety. In some embodiments, the target geneamplification product and the reference gene amplification products arelabeled with different detectable moieties.

In certain embodiments, a plurality of haploid cells (e.g., sperm cells)is obtained from the test subject. The haploid cells are delivered toreaction vessels at a concentration of one cell per vessel. Once sorted,each cell is individually treated to make the genomic DNA of the cellaccessible for the performance of a genetic assay. In some embodiments,the genetic assay involves nucleic acid amplification of the target geneand a reference gene that is present in the cell (e.g., a house-keepinggene) in the same reaction vessel. Statistics can be used to analyzereplicates reactions for the presence or absence of the target gene withrespect to the reference gene, which should always be present. In suchembodiments, where a plurality of haploid cells is assayed, absence ofthe target gene in approximately 50% of the cells from the individualindicates that the individual is a carrier of the null deletionmutation.

In alternative embodiments, a plurality of haploid cells (e.g., spermcells) is obtained from the test subject, and the haploid cells areencapsulated into microdroplets at a concentration of one cell permicrodroplet. The microdroplets can be sorted into reaction vessels at aconcentration of one cell per vessel or plurality of microdroplets canbe contained in a one or more vessels in an aqueous-in-oil emulsion. Insome embodiments, in order to enrich to microdroplets that contain acell, the microdroplets are sorted based on whether the microdropletcontains cell. In some embodiments, the cells are labeled. Themicrodroplets, each containing a haploid cell, is treated to make thegenomic DNA of the cell accessible for the performance of a geneticassay. In some embodiments, the genetic assay involves nucleic acidamplification of the target gene and a reference gene that is present inthe cell (e.g., a house-keeping gene) in the same microdroplet. Theamplification products can be analyzed by detection of the amplificationproducts in each of the microdroplets. In some embodiments, amicrofluidic detection apparatus is employed to scan the droplets forthe target gene and reference gene amplification products. Statisticscan be used to analyze replicates reactions for the presence or absenceof the target gene with respect to the reference gene, which shouldalways be present. In such embodiments, where a plurality of haploidcells is assayed, absence of the target gene in approximately 50% of thecells from the individual indicates that the individual is a carrier ofthe null deletion mutation. Exemplary methods for the microdroplet-basedemulsion amplification and detection from single cells are known and canbe employed in combination with the haploid cell amplification methodsprovided herein (see, e.g., U.S. Pat. Nos. 8,338,166, 8,454,906, Novaket al. (2011) Angew Chem Int Ed Engl. 50(2): 390-395, Clausell-Tormos etal. (2008) Chem. Biol. 15:427-437, Novake et al. (2010) Anal Chem.82(8):3183-90, and Solvas et al. (2001) J. Vis. Exp. (58): e3437).

As described herein, the methods provided are useful for the detectionof the SMN1 silent carrier genotype of SMA in which two copies of theSMN1 gene are located on a single chromosome 5 and no copies of the geneare located on the chromosome 5 homolog. In certain embodiments, aplurality of haploid cells is obtained from the test subject suspectedof having SMA. In such cases, single haploid cells obtained from thesubject are delivered to reaction vessels at a concentration of one cellper vessel. Each cell is individually treated to make the genomic DNA ofthe cell accessible for the performance of a genetic assay for thedetection of the SMN1 gene. In some embodiments, the genetic assayinvolves nucleic acid amplification of a target region of the SMN1 geneand a target region of a reference gene that is present in the cell(e.g., a house-keeping gene) in the same reaction vessel. Statistics canbe used to analyze replicates reactions for the presence or absence ofthe SMN1 gene with respect to the reference gene, which should always bepresent. In such embodiments, where a plurality of haploid cells isassayed, absence of the SMN1 gene in approximately 50% of the cells fromthe individual indicates that the individual is a carrier of the SMN1null deletion mutation.

In alternative embodiments for detection of an individual as a carrierof the SMN1 null deletion mutation, a plurality of haploid cells (e.g.,sperm cells) is obtained from the test subject, and the haploid cellsare encapsulated into microdroplets at a concentration of one cell permicrodroplet. The microdroplets can be sorted into reaction vessels at aconcentration of one cell per vessel or plurality of microdroplets canbe contained in a one or more vessels in an aqueous-in-oil emulsion. Insome embodiments, in order to enrich to microdroplets that contain acell, the microdroplets are sorted based on whether the microdropletcontains cell. In some embodiments, the cells are labeled. Themicrodroplets, each containing a haploid cell, is treated to make thegenomic DNA of the cell accessible for the performance of a geneticassay for the detection of the SMN1 gene. In some embodiments, thegenetic assay involves nucleic acid amplification of a target region ofthe SMN1 gene and a reference gene that is present in the cell (e.g., ahouse-keeping gene) in the same microdroplet. The amplification productscan be analyzed by detection of the amplification products in each ofthe microdroplets. In some embodiments, a microfluidic detectionapparatus is employed to scan the droplets for the target gene andreference gene amplification products. Statistics can be used to analyzereplicates reactions for the presence or absence of the SMN1 gene withrespect to the reference gene, which should always be present. In suchembodiments, where a plurality of haploid cells is assayed, absence ofthe SMN1 gene in approximately 50% of the cells from the individualindicates that the individual is a carrier of the null deletionmutation. Exemplary methods for the microdroplet-based emulsionamplification and detection from single cells are known and can beemployed in combination with the haploid cell amplification methodsprovided herein for detection of the SMN1 null deletion mutation andsilent carrier status (see, e.g., U.S. Pat. Nos. 8,338,166, 8,454,906,Novak et al. (2011) Angew Chem Int Ed Engl. 50(2): 390-395,Clausell-Tormos et al. (2008) Chem. Biol. 15:427-437, Novake et al.(2010) Anal Chem. 82(8):3183-90, and Solvas et al. (2001) J. Vis. Exp.(58): e3437).

In some embodiments, the assay further involves analysis of the genecopy number in diploid cells of the test subject. In some embodiments,genetic analysis of single diploid cells from the test subject isperformed to determine the copy number of the SMN1 and/or SMN2 genes. Insome embodiments, genetic analysis of single diploid cells from the testsubject is performed to confirm that the subject has two copies of theSMN1 gene. In some embodiments, an assay to determine gene copy numberof the SMN1 gene is performed as described in Curet et al. (2007)Neurogenetics 8:271-278. In some embodiments, the diploid cells areblood cells.

In some embodiments, the assay further involves generation of cell linesfrom diploid cells (e.g. from lymphocytes, fibroblasts, stem cells,epithelial cells, etc.) of the test subject. In some embodiments, celllines are generated using standard techniques for cell transformation(e.g., Hahn (2002) Mol. Cells 13(3):351-361; Stabley et al. (2015) Mol.Gen. Genomic Med. 3(4) 248-257). In some embodiments, the transformedcell lines are employed for analysis of gene copy number of the SMN1and/or SMN2 genes. In some embodiments, genetic analysis of thetransformed cell lines is performed to confirm that the subject has twocopies of the SMN1 gene.

In some embodiments, the assay further involves generation of cell linesfrom diploid cells of the test subject identified as having a SMN1 genesilent carrier genotype. Generation of cell lines provides a long termrecord of an individual with this rare genotype. An immortal cell lineprovides an unlimited volume of sample from such an individual withoutany additional sample draws. Such cells lines can be employed toidentify sequence markers unique to “silent carrier” founder alleles.

In some embodiments, the methods further comprise sequencing of thetarget genes, e.g., SMN1 and SMN2 genes, or one or more portionsthereof. In some embodiments, the target gene amplification products aresequenced. In some embodiments, the target gene amplification productsgenerated by PCR in aqueous-oil microdroplets are sequenced. Anysuitable method for sequencing nucleic acids can be employed. In someembodiments, next generation sequencing is employed.

In some embodiments, the methods further involve the generation of areport based on the results of the assay. In some embodiments, themethods further involve determining the risk of producing offspring withSMA based on the results of the assay.

Target and Reference Genes

The methods described herein can be employed for the detection of a nulldeletion in a target gene. In particular embodiments, the target gene isone where at least one copy of the gene is deleted on one chromosome andpresent in multiple copies (e.g., 2, 3, 4 or more copies) on thehomologous chromosome or other location(s) in a silent carrier. Inparticular embodiments, the target gene is the SMN1 gene.

For practice of the methods provided herein, the absence of the targetgene is determined by the absence of a nucleic acid amplificationproduct relative to a reference nucleic acid amplification product froma selected reference gene, where the target gene and the reference geneare amplified in the same reaction vessel. Exemplary reference genes foruse in the methods provided include, but are not limited to, cysticfibrosis transmembrane transregulator (CFTR), Glyceraldehyde-3-phosphatedehydrogenase (GAPDH), Beta-2-microglobulin (B2M), Hydroxymethyl-bilanesynthase (HMBS), Hypoxanthine phosphoribosyl-transferase 1 (HPRT1),Ribosomal protein L13a (RPL13A), Succinate dehydrogenase complex,subunit A (SDHA), TATA box binding protein (TBP), Ubiquitin C (UBC),Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase binding tophosphorylated activation protein, zeta polypeptide (YWHAZ),peroxiredoxin-6 (PRDX6), Alpha-adducin (ADD1), Major HistocompatibilityComplex, Class I, A (HLA-A), RAD9A, Rho Guanine Nucleotide ExchangeFactor (GEF) 7 (ARHGEF7), Eukaryotic Translation Initiation Factor 2B,Subunit 2 Beta (EIF2B2), 26S Proteasome Non-ATPase Regulatory Subunit 7(PSMD7), Branched Chain Amino-Acid Transaminase 2 (BCAT2), and ATPSynthase Subunit O (ATP5O). In particular embodiments, the referencegene for use in the methods provided herein is the CFTR gene.

Subjects for Testing and Sample Acquisition

Generally, the methods provided herein are employed to determine thesilent carrier status in a mammal (e.g., primate, rabbit, dog, cat,sheep and pig). In particular embodiments, the subject is a humanpatient.

In certain instances, selection of subjects for testing for silentcarrier status for a particular target gene is based on multiplefactors. In some embodiments, the subject is selected for testing basedon the prevalence of the deletion in the general population or aparticular ethnic group. In some embodiments, the subject is selectedfor testing based on a confirmed or suspected family history of adisease associated with the target gene. In some embodiments, thesubject is selected for testing based on a confirmed or suspected familyhistory of SMA. In certain instances, a subject is selected for testingfor silent carrier status based on the recommendation of a licensedphysician or as a part of a genetic counseling procedure or program.

In certain instances, a subject selected for testing is suspected ofhaving a deletion a deletion of the SMN1 gene on one chromosome 5homolog and two or more copies of the SMN1 gene on the other chromosome5 homolog.

In particular embodiments, natural haploid cells are employed in theassay. In such instances, standard methods for obtaining male or femalegametes as appropriate for the particular subject can be employed.

In particular embodiments, induced haploid cells derived from adult stemcells are employed in the assay. In such instances, any source of stemcells from the subject can be used. Adult stem cells can be obtainedfrom a variety of organs and tissues, including, but not limited tobrain, bone marrow, peripheral blood, blood vessels, skeletal muscle,skin, teeth, heart, gut, liver, ovarian epithelium, and testis. Incertain instances, the adult stem cells are treated to inducepluripotency of the stem cells (e.g., generate an induced pluripotentstem cell (iPSC)). For example, in certain instances, adult stem cellscan be modified to express one or more genes that induce pluripotency,such as, for example, Oct4, Sox2, cMyc, and/or Klf4. Once a pluripotentcell is generated, the cell can be treated to induce meiosis to generatean induced haploid cell (see, e.g., Eguizabal et al. (2011) Stem Cells29:1186-1195).

Single Cell Sorting Methods

Any suitable method for sorting individual haploid cells into separatereaction vessels for analysis can be used in the methods provided.Exemplary cell sorting methods include, but are not limited to, dilutionsorting, droplet based microfluidics, flow cytometry, fluorescenceactivated cell sorting (FACS), magnetic activated cell sorting (MACS),laser-assisted cell picking, micropatterning on controlled patches ofextracellular matrix (ECM) or other ligands or microfluidic chipsorting. In some embodiments, the cells are seeded into a reactionvessel at a concentration of one cell per well. Methods for sorting ofsingle cells and seeding of single cells into a variety of reactionvessels for genomic analysis are known in the art and include, forexample, methods as described in U.S. Pat. No. 6,673,542 and U.S. PatentPub. No. 2011/0237445.

In certain embodiments, the haploid cells are labeled with a suitabledye (e.g., Hoechst 33342) to assist the haploid cell sorting method. Insuch methods, the haploid cells are contacted with the dye for apredetermined length of time to allow for labeling of the haploid cells.The labeled haploid cells are then subjected to the selected cellsorting method.

Cells can be sorted into any suitable reaction vessel appropriate forperformance of the methods provided herein. In some embodiments, thecells are sorted in a suitable reaction vessel appropriate for thehybridization of a gene-specific probe. In some embodiments, the cellsare sorted in a suitable reaction vessel appropriate for nucleic acidamplification. Exemplary reaction vessels include, but are not limitedto, multiwell plates, microtiter plates, reaction grid slides (e.g.,AmpliGrid slides and chemically structured glass slides containinghydrophilic anchor spots each framed by a hydrophobic ring) and PCRtubes. In particular embodiments, the single cells are sorted into amultiwell plate such as a 96-, 384-, 1536- or greater multiwell plate.In some embodiments, the placement of single cells into the reactionvessels is confirmed by visual or automated inspection under amicroscope. In some embodiments, the placement of single cells into thereaction vessels is confirmed by addition of cell specific dye or probe.In some embodiments, the single haploid cells are labeled prior to cellsorting and confirmation of cell sorting is confirmed by detection ofthe labeled cells. For example, in some embodiments, detection of thestrength of the signal, such as fluorescent signal, is indicative thenumber of cells per well.

In some embodiments, the cells are seeded into reaction vessels at aconcentration of a single cell per reaction vessel for lysis of thecells, and the nucleic acid amplification reaction is performed in thesame reaction vessel. In some embodiments, the cells are seeded intoreaction vessels at a concentration of a single cell per reaction vesselfor lysis of the cells, and the nucleic acid amplification reaction isperformed in a different reaction vessel (i.e., the genomic DNA sampleis transferred to a new reaction vessel for the nucleic acidamplification reaction).

In some embodiments, the cells are encapsulated individually inmicrodroplets. A microdroplet generally includes an amount of a firstsample fluid in a second carrier fluid. Any technique known in the artfor forming droplets may be used with methods of the invention. Anexemplary method involves flowing a stream of the sample fluidcontaining the target material (e.g., a haploid cell) such that itintersects two opposing streams of flowing carrier fluid. The carrierfluid is immiscible with the sample fluid. Intersection of the samplefluid with the two opposing streams of flowing carrier fluid results inpartitioning of the sample fluid into individual sample dropletscontaining the target material. The carrier fluid may be any fluid thatis immiscible with the sample fluid. An exemplary carrier fluid is oil.In certain embodiments, the carrier fluid includes a surfactant.

In some embodiments, a microfluidic device is used to generate singlecell emulsion droplets. The microfluidic device ejects single cells inaqueous reaction buffer into a hydrophobic oil mixture. The device cancreate thousands of emulsion microdroplets per minute. After theemulsion microdroplets are created, the device ejects the emulsionmixture into a trough. The mixture can be pipetted or collected into astandard reaction tube for lysis and/or thermocycling. In someembodiments the microdroplets are seeded into individual reactionvessels (e.g. a microtiter plate, a microchip or reaction grid slide).

In some embodiments, the microdroplets are sorted to enrich formicrodroplets carrying a single haploid cell. In some embodiments, themicrodroplets carrying single haploid cells are sorted based ondifferences the light refractory properties of the microdropletscarrying single haploid cells from empty microdroplets and/ormicrodroplets carrying more than one haploid cell. In some embodiments,the haploid cells are labeled with a suitable dye (e.g., Hoechst 33342)to assist the cell sorting method. In such methods, the haploid ells arecontacted with the dye for a predetermined length of time to allow forlabeling of the haploid cells. The microdroplets carrying labeledhaploid cells are then subjected to the selected cell sorting method.

Droplets can be generated having an average diameter of about, less thanabout, or more than about, or at least about 0.001, 0.01, 0.05, 0.1, 1,5, 10, 20, 30, 40, 50, 60, 70, 80, 100, 120, 130, 140, 150, 160, 180,200, 300, 400, or 500 microns. Droplets can have an average diameter ofabout 0.001 to about 500, about 0.01 to about 500, about 0.1 to about500, about 0.1 to about 100, about 0.01 to about 100, or about 1 toabout 100 microns. Microfluidic methods of producing emulsion dropletsusing microchannel cross-flow focusing or physical agitation are knownto produce either monodisperse or polydisperse emulsions. The dropletscan be monodisperse droplets. The droplets can be generated such thatthe size of the droplets does not vary by more than plus or minus 5% ofthe average size of the droplets. In some cases, the droplets aregenerated such that the size of the droplets does not vary by more thanplus or minus 2% of the average size of the droplets. A dropletgenerator can generate a population of droplets from a single sample,wherein none of the droplets vary in size by more than plus or minusabout 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%,6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of the average size of thetotal population of droplets.

Microfluidic systems and devices have been described in a variety ofcontexts, typically in the context of miniaturized laboratory (e.g.,clinical) analysis. Other uses have been described as well. For example,International Patent Application Publication Nos. WO 01/89788; WO2006/040551; WO 2006/040554; WO 2004/002627; WO 2008/063227; WO2004/091763; WO 2005/021151; WO 2006/096571; WO 2007/089541; WO2007/081385 and WO 2008/063227.

Custom microfluidics devices for single-cell analysis are routinelymanufactured in academic and commercial laboratories (Kintses et al.(2010) Current Opinion in Chemical Biology 14:548-555). For example,chips may be fabricated from polydimethylsiloxane (PDMS), plastic,glass, or quartz. In some embodiments, fluid moves through the chipsthrough the action of a pressure or syringe pump. Single cells can evenbe manipulated on programmable microfluidic chips using a customdielectrophoresis device (Hunt et al. (2008) Lab Chip 8:81-87). In oneembodiment, a pressure-based PDMS chip comprised of flow-focusinggeometry manufactured with soft lithographic technology is used(Dolomite Microfluidics (Royston, UK)) (Anna et al. (2003) AppliedPhysics Letters 82:364-366). The stock design can typically generate10,000 aqueous-in-oil microdroplets per second at size ranges from10-150 μm in diameter. In some embodiments, the hydrophobic phase willconsist of fluorinated oil containing an ammonium salt ofcarboxy-perfluoropolyether, which ensures optimal conditions formolecular biology and decreases the probability of droplet coalescence(Johnston et al. (1996) Science 271:624-626). To measure periodicity ofcell and droplet flow, images can be recorded at 50,000 frames persecond using standard techniques, such as a Phantom V7 camera or FastecInLine (Abate et al. (2009) Lab Chip 9:2628-31).

The microfluidic system can optimize microdroplet size, input celldensity, chip design, and cell loading parameters such that greater than98% of droplets contain a single cell. Three common methods forachieving such statistics are: (i) extreme dilution of the cellsolution; (ii) fluorescent selection of droplets containing singlecells; and (iii) optimization of cell input periodicity. For eachmethod, the metrics for success include: (i) encapsulation rate (i.e.,the number of drops containing exactly one cell); (ii) the yield (i.e.,the fraction of the original cell population ending up in a dropcontaining exactly one cell); (iii) the multi-hit rate (i.e., thefraction of drops containing more than one cell); (iv) the negative rate(i.e., the fraction of drops containing no cells); and (v) encapsulationrate per second (i.e., the number of droplets containing single cellsformed per second).

In some embodiments, single cell emulsions are generated by extreme celldilution. Under disordered conditions, the probability that amicrodroplet will contain k cells is given by the Poisson distribution:f(k;λ)=(λ^(k) e ^(−λ))/k!,

where e is the natural logarithm and the expected number of occurrencesin the interval is λ. Thus, for P(k=1)≈0.98, the cell solution must beextremely dilute, such that A 0.04 and only 3.84% of all drops contain asingle cell.

In some embodiments, a simple microfluidic chip with a drop-makingjunction is used, such that an aqueous stream flows through a 10 μmsquare nozzle and dispenses the aqueous-in-oil emulsion mixtures into areservoir. The emulsion mixture can then be pipetted from the reservoirand thermocycled in standard reaction tubes, microtiter plates,microchips or reaction grid slides. This method will produce predictablyhigh encapsulation rates and low multi-hit rates, but a lowencapsulation rate per second. A design that can achieve filled dropletthroughput of 1000 Hz is capable of sorting up to 10⁶ cells in less than17 minutes.

In some embodiments, fluorescence techniques can also be used to sortmicrodroplets with particular emission characteristics (Baroud et al.(2007) Lab Chip 7:1029-1033; Kintses et al. (2010) Current Opinion inChemical Biology 14:548-555). In these studies, chemical methods areused to stain cells. In some embodiments, autofluorescence is used toselect microdroplets that contain cells. A fluorescent detector reducesthe negative rate resulting from extreme cell dilution. A microfluidicdevice can also be equipped with a laser directed at a “Y” sortingjunction downstream of the cell encapsulation junction. The Y junctionhas a “keep” and a “waste” channel. A photomultiplier tube is used tocollect the fluorescence of each drop as it passes the laser. Thevoltage difference is calibrated between empty drops and drops with atleast one cell. Next, when the device detects a droplet that contains atleast one cell, and electrodes at the Y sorting junction create a fieldgradient by dielectrophoresis (Hunt et al. (2008) Lab on a Chip 8:81-87)and push droplets containing cells in to the keep channel. Themicrofluidic device uses extreme cell dilution to control the multi-hitrate and fluorescent cell sorting to reduce the negative rate.

In some embodiments, input cell flow is aligned with droplet formationperiodicity, such that greater than 98% of droplets contain a singlecell (Edd et al. (2008) Lab Chip 8:1262-1264; Abate et al. (2009) LabChip 9:2628-31). In these microfluidic devices, a high-densitysuspension of cells is forced through a high aspect-ratio channel, suchthat the cell diameter is a large fraction of the channel's width. Thechip is designed with a 27 μm×52 μm rectangular microchannel that flowscells into microdroplets at >104/min (Edd et al. (2008) Lab Chip8:1262-1264). A number of input channel widths and flow rates are testedto arrive at an optimal solution.

In some embodiments, cells with different morphology behave differentlyin the microchannel stream of the microfluidic device, confoundingoptimization of the technique when applied to clinical biologicalsamples. To address this issue, in some embodiments, a field gradientperpendicular to the microchannel by dielectrophoresis is induced.Dielectrophoresis pulls the cells to one side of the microchannel,creating in-channel ordering that is independent of cell morphology.This method requires substantial optimization of charge and flow rateand a more complicated chip and device design, so this method may benecessary if existing methodologies fail to perform for certain celltypes.

In some embodiments, the methods of the invention use single cells inreaction containers, rather than emulsion droplets. Examples of suchreaction containers include 96 well plates, 0.2 mL tubes, 0.5 mL tubes,1.5 mL tubes, 384-well plates, 1536-well plates, etc.

Preparation of Genomic DNA

Preparation of a genomic DNA sample from haploid cells for a geneticassay, such as nucleic acid amplification, typically involves lysis ofthe cells to expose the genomic DNA. Any suitable lysis buffer for thepreparation of genomic DNA from cells can be used. In certain instances,particular haploid cells, such as sperm cells, are resistant toconventional lysis procedures. Accordingly, in certain embodiments, themethods for preparation of a genomic DNA sample involve lysis of thecells using one or more suitable enzymes (e.g. a protease, such asproteinase K).

In some embodiments, the haploid cells are lysed in an alkaline lysissolution (e.g. a potassium hydroxide alkaline lysis solution) ordetergent solution, e.g. Tween 20. In some embodiments, the lysissolution contains enzyme to assist in lysis, such as, for example, aprotease (e.g., proteinase K). In some embodiments, the lysis solutionalso contains one or more additional components, such as a redoxstabilization reagent (e.g., dithiothreitol (DDT)), a chelating agent(e.g., EDTA) or a buffering agent.

In some embodiments, where the haploid cells are encapsulated inmicrodroplets, the lysis buffer is introduce at the time of cellencapsulation using a co-flow drop maker to prevent premature rupture ofthe cells.

Nucleic Acid Amplification and Detection

Following preparation of the genomic DNA, a genetic assay is performedto detect the target gene null allele. In particular embodiments of themethods provided, the target gene null allele is detected by nucleicacid amplification, for example, by polymerase chain reaction (PCR). Insome embodiments, the region that overlaps or contains the deletion inthe target gene is amplified from the genomic DNA sample from the singlehaploid cell from the subject. In samples that have the deletion, thetarget gene region will not be amplified. To ensure that the genomic DNAsample was present in the reaction vessel and that the conditions fornucleic acid amplification were suitable, a reference gene is alsoamplified in the same amplification reaction as the target gene.Accordingly, success or failure of the target gene amplification isassessed relative to the amplification of the reference gene.

For any particular subject that is a silent carrier for the target genenull allele, approximately 50% of the haploid cells produced by thesubject will have the null allele. Accordingly, 50% of the haploid cellstested from the subject using the methods provided herein will fail toamplify the target gene relative to the reference gene, indicating thatthe target gene has the deletion in the haploid cell. Accordingly,multiple amplification reactions are performed (i.e., multiple haploidcells from the subject tested) in order to confirm that the deletion ispresent. In some embodiments 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 500, 1000, 5000 or more amplification reactions areperformed, where each amplification reaction represents a single haploidcell.

Exemplary reaction vessels for nucleic acid amplification include, butare not limited to, multiwell plates, microtiter plates, microchips,reaction grid slides (e.g., AmpliGrid slides and chemically structuredglass slides containing hydrophilic anchor spots each framed by ahydrophobic ring) and PCR tubes.

Exemplary nucleic acid amplification reaction mixtures for the practiceof the methods contain a DNA template (e.g., a genomic DNA sampleobtained from a single haploid cell), at least one oligonucleotideprimer set specific for a target gene (e.g., for amplification of atarget region of a target gene), at least one oligonucleotide primer setspecific for a reference gene (e.g., for amplification of a targetregion of the reference gene), a thermostable DNA polymerase,deoxynucleoside triphosphates (dNTPs), Mg²⁺, and a suitable buffer.

In exemplary embodiments, the amplification reaction mixture comprisesabout, more than about, or less than about 1, 5, 10, 15, 20, 30, 50,100, or 200 mM Tris. In some embodiments, the amplification reactionmixture comprises potassium chloride at a concentration about, more thanabout, or less than about 10, 20, 30, 40, 50, 60, 80, 100, 200 mM. Insome embodiments, the amplification reaction mixture comprises about 15mM Tris and 50 mM KCl. In some embodiments, the amplification reactionmixture comprises deoxyribonucleotide triphosphate molecules, includingdATP, dCTP, dGTP, dTTP, in concentrations of about, more than about, orless than about 50, 100, 200, 300, 400, 500, 600, or 700 μM each. Insome embodiments, magnesium chloride or magnesium acetate (MgCl₂) isadded to the amplification reaction mixture at a concentration of about,more than about, or less than about 1.0, 2.0, 3.0, 4.0, or 5.0 mM. Insome embodiments, the amplification reaction mixture comprises MgCl₂ ata concentration about 3.2 mM. In some embodiments, the amplificationreaction mixture comprises magnesium acetate or magnesium is used. Insome embodiments, magnesium sulfate. In some embodiments, theamplification reaction mixture comprises a non-specific blocking agent,such as BSA or gelatin from bovine skin, wherein the gelatin or BSA ispresent in a concentration range of approximately 0.1-0.9% w/v. Otherpossible blocking agents can include betalactoglobulin, casein, drymilk, or other common blocking agents. In some cases, preferredconcentrations of BSA and gelatin are about 0.1% w/v

Exemplary polymerase enzymes for nucleic acid amplification include, butare not limited to, thermostable DNA polymerases, such as Thermusthermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNApolymerase, Thermotoga neopalitana (Tne) DNA polymerase, Thermotogamaritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT™) DNApolymerase, Thermus eggertssonii (Teg) DNA polymerase, Pyrococcusfuriosus (Pfu) DNA polymerase, DEEPVENT. DNA polymerase, Pyrococcuswoosii (Pwo) DNA polymerase, Pyrococcus sp KDD2 (KOD) DNA polymerase,Bacillus sterothermophilus (Bst) DNA polymerase, Bacillus caldophilus(Bea) DNA polymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase,Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus (Tfl/Tub)DNA polymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus(DYNAZYME) DNA polymerase, Methanobacterium thermoautotrophicum (Mth)DNA polymerase, mycobacterium DNA polymerase (Mtb, Mlep), or mutants,variants or derivatives thereof. In some embodiments, the polymerase isa hot-start polymerase, such as a hot start Taq polymerase. In someembodiments, the polymerase is a chemically modified hot-startpolymerase or an antibody modified hot start polymerase.

Standard methods for nucleic acid amplification of nucleic acid fromgenomic DNA obtained from a single cell are known in the art and can beemployed in the methods provided herein (see, e.g., U.S. Patent Pub. No.2011/0237445). In an exemplary protocol, nucleic acid amplificationcomprises, in general steps, (a) contacting each nucleic acid strandtemplate with four different nucleotide triphosphates and oneoligonucleotide primer pair for each different specific sequence beingamplified, wherein each primer of the primer pair is selected to besubstantially complementary to different strands of each specificsequence, such that the extension product synthesized from one primer,when it is separated from its complement, can serve as a template forsynthesis of the extension product of the other primer, said contactingbeing at a temperature which promotes hybridization of each primer toits complementary nucleic acid strand; (b) contacting each nucleic acidstrand, at the same time as or after step (a), with a thermostable DNApolymerase such as from Thermus aquaticus which enables combination ofthe nucleotide triphosphates to form primer extension productscomplementary to each strand of each nucleic acid; (c) maintaining themixture from step (b) at an effective temperature for an effective timeto promote the activity of the enzyme, and to synthesize, for eachdifferent sequence being amplified, an extension product of each primerwhich is complementary to each nucleic acid strand template, but not sohigh as to separate each extension product from its complementary strandtemplate; (d) heating the mixture from step (c) for an effective timeand at an effective temperature to separate the primer extensionproducts from the templates on which they were synthesized to producesingle-stranded molecules, but not so high as to irreversibly denaturethe enzyme; (e) cooling the mixture from step (d) for an effective timeand to an effective temperature to promote hybridization of each primerto each of the single-stranded molecules produced in step (d); and (f)maintaining the mixture from step (e) at an effective temperature for aneffective time to promote the activity of the enzyme and to synthesize,for each different sequence being amplified, an extension product ofeach primer which is complementary to each nucleic acid strand templateproduced in step (d), but not so high as to separate each extensionproduct from its complementary strand template wherein the effectivetime and temperatures in steps (e) and (f) may coincide (steps (e) and(f) are carried out simultaneously), or may be separate. Steps (d)-(f)may be repeated until the desired level of sequence amplification isobtained.

In some embodiments, where the lysed haploid cells are encapsulated inmicrodroplets, the amplification reaction mixture is introduced bydilution of the microdroplets by droplet merger and/or dropletpicoinjection of the amplification reagents.

In some embodiments, the amplification reaction is carried out inmicrodroplets by performing digital PCR, such as microfluidic-baseddigital PCR or droplet digital PCR. In some embodiments, thermal cyclingis accomplished in a single vessel (e.g. tube, microtiter well, amicrochip or reaction grid slide) containing a plurality ofmicrodroplets or as a continuous flow of the microdroplets through amicrofluidic channel through defined temperature zones (see, e.g., USPatent Pub. 2009/0042737).

In some cases, a target region for amplification is about, more thanabout, or less than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000,11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000,or 20,000 bases or base-pairs in length. In some cases, a target foramplification is about 10 to about 100, about 100 to about 200, about100 to about 300, about 100 to about 400, about 100 to about 500, about100 to about 600, about 100 to about 700, about 100 to about 800, about100 to about 900, about 100 to about 1000, about 1000 to about 2000,about 1000 to about 5000, or about 1000 to about 10,000 bases orbase-pairs in length.

The length of the forward primer and the reverse primer can depend onthe sequence of the target polynucleotide and the target locus. Forexample, the length and/or Tm of the forward primer and reverse primercan be optimized. In some case, a primer can be about, more than about,or less than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, or 60 nucleotides in length. In some cases, a primer is about 15 toabout 20, about 15 to about 25, about 15 to about 30, about 15 to about40, about 15 to about 45, about 15 to about 50, about 15 to about 55,about 15 to about 60, about 20 to about 25, about 20 to about 30, about20 to about 35, about 20 to about 40, about 20 to about 45, about 20 toabout 50, about 20 to about 55, or about 20 to about 60 nucleotides inlength.

In some embodiments, primers for amplification within the amplificationreaction mixture can have a concentration of about, more than about, orless than about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,1.2, 1.5, 1.7, or 2.0 μM. Primer concentration within the aqueous phasecan be about 0.05 to about 2, about 0.1 to about 1.0, about 0.2 to about1.0, about 0.3 to about 1.0, about 0.4 to about 1.0, or about 0.5 toabout 1.0 μM. The concentration of primers can be about 0.5 μM. Amenableranges for target nucleic acid concentrations in PCR are between about 1pg and about 500 ng

In an exemplary assay according to methods provided herein, the nucleicacid amplification reaction contains an oligonucleotide primer pair thatis an oligonucleotide primer set for amplification of a target region ofthe target gene (e.g., exon 7 of SMN1 or a portion thereof) and anoligonucleotide primer set specific for the reference gene. Exemplaryprimers for the amplification of a target gene (e.g., an SMN1 targetgene) and a reference gene (e.g., a CFTR reference gene) are provided.In some embodiments, for amplification of the target region of SMN1, atleast one of the primers of the primer pairs distinguishes between SMN1and SMN2. SMN1 and SMN2 differ by a single nucleotide difference (C/T)at position 6 in exon 7 of SMN1 and SMN2. In some embodiments, theallele specific primer is a forward primer distinguishes between SMN1and SMN2, having a C or T, respectively, at the 3′ end of the primercorresponding to position 6 in exon 7 of SMN1 and SMN2. In someembodiments, a mismatch T→G also added at the −3 position from the 3′end of both SMN1 and SMN2 forward primers which aids in allelespecificity.

In some embodiments, the forward primer for amplification of the targetregion of SMN1 has the sequence SEQ ID NO: 1. In some embodiments, thereverse primer for amplification of the target region of SMN1 has thesequence SEQ ID NO: 3. In some embodiments, the forward primer foramplification of the target region of SMN2 has the sequence SEQ ID NO:2. In some embodiments, the reverse primer for amplification of thetarget region of SMN2 has the sequence SEQ ID NO: 3.

In some embodiments, the forward primer for amplification of a CFTRreference gene has the sequence SEQ ID NO: 4. In some embodiments, thereverse primer for amplification of a CFTR reference gene has thesequence SEQ ID NO: 5.

In some embodiments, one or more oligonucleotide primers in the nucleicacid amplification reaction are labeled. In some embodiments, theoligonucleotide primers are labeled with a detectable moiety, such as aradioactive moiety, a fluorescent moiety, or a dye molecule. In someembodiments, the composition comprises a dual labeled fluorescenceenergy transfer (FRET) probe. In particular embodiments, at least oneprimer of the oligonucleotide primer set specific for the target geneand at least one primer of the oligonucleotide primer set specific forthe reference gene is labeled.

In some embodiments, the presence or absence of the target gene andreference gene amplification products are detected following the nucleicacid amplification reaction. Any suitable method for the detection of anamplification product, such as by gel electrophoresis or labeled nucleicacid probes, can be employed.

In some embodiments, a quantitative PCR method is employed to monitorthe amplification of target gene and the reference gene in each nucleicacid reaction (e.g. a comparative Ct method). In such methods, at leastone oligonucleotide primer of each primer pair specific for the targetgene or the reference gene is labeled with a different detectablemoiety, which allows detection of the different amplification productswithin the same reaction vessel.

In particular embodiments, the methods further comprise confirmation ofthe gene copy number in the test subject. In some embodiments, gene copynumber is determined by nucleic acid amplification from a genomic DNAsample isolated from a plurality of diploid cells or a plurality ofhaploid cells from the subject using a quantitative PCR analysis method(e.g. RT PCR). In such methods, the copy number is determined bycomparative Ct against the reference gene.

In some embodiments, one or more oligonucleotide primers in the nucleicacid amplification reaction comprise additional nucleic acid sequencesfor identification of the amplification products and/or to assist insubsequent manipulation or analysis. For example, in some embodimentsone or more oligonucleotide primers in the nucleic acid amplificationreaction comprise a unique nucleic acid barcode. In some embodiments,one or more oligonucleotide primers in the nucleic acid amplificationreaction comprise an adapter sequence for further amplification, forannealing of a sequencing primer, anchoring of the amplification productto a solid support such as a microbead. In some embodiments, one or moreoligonucleotide primers is linked to a solid support such as amicrobead.

Data Analysis

Following nucleic acid amplification and detection of the amplifiedproducts, the number samples that contain each amplification product,the reference gene amplification product and/or the target geneamplification product is counted. Determination of silent carrier statusis determined based on the absence of the target gene amplificationproduct in approximately 50% of the samples that contain a referencegene amplification product. In some embodiments, the ratio of the targetgene amplification product to the reference gene amplification productis determined. For example, a ratio of approximately 0.5 for the targetgene amplification product to the reference gene amplification productis indicative of a silent carrier.

For individuals that are not silent carriers for SMN1 gene deletion andcontain two copies of the SMN1 gene, it is expected that the ratio ofthe target gene amplification product to the reference geneamplification product is about 1. In some embodiments, samples areselected for further analysis if the ratio of the target geneamplification product to the reference gene amplification productdeviates significantly from about 1. In some embodiments, a significantdeviation of the ratio of the target gene amplification product to thereference gene amplification product from about 1 indicates that theindividual is a silent carrier of the SMN1 null allele. Accordingly, insome embodiments, potential silent carriers are selected if the ratio ofthe target gene amplification product to the reference geneamplification product is at or below a threshold level. The thresholdlevel can be determined by as appropriate statistical method, forexample a single value t-test. In some embodiments, the threshold levelis at or below 0.8. In some embodiments, the threshold level is at orbelow 0.75.

It is understood that the methods provided herein can be performed withthe assistance of one or more automated devices or computer modules. Forexample, procedures for cell seeding, preparation of genomic DNA,dispensing, mixing, removal and/or transfer of reagents to or fromreaction vessels, thermocycling for nucleic amplification, detection andquantitation of amplification products, analysis of data, and generationof a report can be automated in part or entirely with the assistance ofone or more automated devices or computer modules.

Kits

In some embodiments, provided are kits for the practice of the methodsprovided herein. In some embodiments, the kits contain one or morereagents for the performance of an amplification reaction for the targetgene and a reference gene, and optionally, instructions for use. In someembodiments, the contain reaction vessels, such as microtiter plates,microchips or reaction grid slides and/or suitable containers for thepractice of the methods provided herein.

In some embodiments, provided are a microtiter plates containing one ormore reagents for the performance of an amplification reaction for thetarget gene and a reference gene. In some embodiments, the one or morereagents are lyophilized in the microtiter plate. In some embodiments,the lyophilized reagents are reconstituted in an appropriate bufferprior to use. For example, the lyophilized reagents are reconstituted inan appropriate buffer prior to addition of the genomic DNA sample. Insome embodiments, the microtiter plate contains a buffering agent. Insome embodiments, the buffering agent is selected from the among Tris,MOPS, HEPES, TAPS, Bicine, Tricine, TES, PIPES, IVIES. In someembodiments, the buffering agent is Tris. In some embodiments, themicrotiter plate contains a polymerase and a polymerase stabilizingagent, such as a non-ionic detergent, a zwitterionic compound, acationic ester compound, a polymer, BSA, or a polysaccharide. In someembodiments the microtiter plate contains at least one dNTP. In someembodiments, the microtiter plate contains an oligonucleotide primer setfor amplification of the reference gene, the target gene, or both thereference gene and the target gene.

EXAMPLES Example 1 Method

Frozen human semen specimens (Bioreclamation IVT) were thawed andcounted on a TC20 Automated Cell Counter (BioRad). Prior to counting,specimens were incubated for 30 minutes at 37° C., vortexed for aminimum of 30 seconds and diluted 1:1 with TE to ensure a single cellsuspension. The resulting counts were then used for the genotyping andsingle sperm assays. For the SMN1 and SMN2 genotyping assay, DNA fromthe sperm specimens was extracted using a modified Puregene manualextraction protocol (Qiagen). For the single cell sperm assay, eachspecimen was individually counted on the TC20 cell counter and dilutedto a final concentration of 0.8 cells/μl and 0.4 cells/W.

Sperm lysis was performed in a 96 well PCR plate, to which 1W of thediluted donor sperm specimens was added to 5 W of lysis buffer (0.1 MDDT, 10 mM EDTA, 0.4 M KOH, and 10% Roche recombinant PCR gradeproteinase K) for a total volume of 6W/well. Each specimen had 48replicate wells for the 0.8 cells/μl and 0.4 cells/μl finalconcentrations. Once the lysis was completed, each plate was prepped forthe quantitative PCR assay that was slightly modified from the standardoperating procedure to account for a 50W total reaction volume. SMN1probes and primers (100 μM concentration) were added to TaqMan FastAdvanced Master Mix (Life Technologies) and run as a comparative CTexperiment on a ViiA 7 Real Time PCR System for 60 cycles. Specimenswere analyzed using ViiA 7 software where SMN1 and a reference controlprobe positive targets were identified and quantified. Each spermspecimen was diluted to 0.8 and 0.4 cells per well and tested for SMN1as well as a reference gene to confirm the presence of a single cell.Each well was counted and the values for the reference gene and SMN1were compared.

For SMN1 and SMN2 amplification, oligonucleotide primers were designedto amplify exon 7 of each gene as described in Curet et al. (2007)Neurogenetics 8:271-278. The SMN forward primers distinguish betweenSMN1 and SMN2 by ending on the nucleotide difference (C/T) at position 6in exon 7. A mismatch T→G added at the −3 position from the 3′ end ofboth SMN1 and SMN2 forward primers aids in allele specificity.

-ex7F-3g: (SEQ ID NO: 1) 5′-TTCCTTTATTTTCCTTACAGGGTGTC-3′ SMN2-ex7F-3g:(SEQ ID NO: 2) 5′-TTCCTTTATTTTCCTTACAGGGTGTT-3′ SMN-ex7R (SEQ ID NO: 3)5′-GCTGGCAGACTTACTCCTTAATTTAA-3′ CFTR-F (SEQ ID NO: 4)5′-TAGGAAGTCACCAAAGCAGTACAGC-3′ CFTR-R (SEQ ID NO: 5)5′-AGCTATTCTCATCTGCATTCCAATG-3′

Results:

A total of 46 African American males were screened for SMA carrierstatus using the single spermatozoa qPCR assay (FIG. 2). Poor qualityspecimens, including low counts and contamination, resulted ininconsistent cell counts and significant qPCR reaction failure [7/46(15%)]. All reliable specimens had at least 2 copies of the SMN1 genedetected by a traditional dosage assay using DNA extracted from the samesemen specimen (Data not shown). No traditional SMA carriers (a singlecopy of SMN1) were identified in this initial data set.

A single specimen (DS11) statistically deviated (single value t-test pvalue=0.0014) from the expected SMN1 to reference gene ratio of theaverage ratio of two copy individuals with a value of 0.729+/−SD 0.66(FIG. 3). A SMN1 to reference gene ratio of approximately 0.5 suggests acarrier specimen where half of the sperm cells are null for SMN1, with arisk of passing on the disease allele of 50%. An observed genotype of 2copies of SMN1 and 50% null spermatozoa are indicative of a 2+0 genotypeor silent carrier.

The DS11 specimen was retested by qPCR to determine the dosage of SMN1and SMN2 genes and with the single cell assay expanded to two fullplates at two different dilutions (n=192 total wells). The genotype ofspecimen DS11 was confirmed to have two copies of SMN1 and two copies ofSMN2. The SMN1 to reference gene ratio result was 0.589+/−SD 0.024(single value t-test p value=2.2×10⁻⁴), confirming the 2+0 genotype andthe initial silent carrier result (FIG. 4A). The specimen was sequencedby Sanger methodologies for SMN1 and non-specific sequencing for SMN1and the homolog SMN2. Non-specific sequencing of the two homologousgenes showed an approximate 50/50 (C/T) ratio at the +6 position of exon7, indicative of an equal number of copies of the two genes (FIG. 4B).Specific sequencing of the SMN1 gene did not reveal any sequencevariants under the qPCR probe or primer sites that may result in alleledropout or decreased probe affinity (FIG. 4C).

The results of this study support the use of a single cell spermatozoaqPCR assay to identify silent SMA carriers in males, eliminating theresidual risk of traditional gene dosage methods. In combination withspecific sequencing of the SMN1 gene, this novel assay is capable ofidentifying 100% of all male SMA carriers resulting from deletion ormutation of the SMN1 locus.

The examples and embodiments described herein are for illustrativepurposes only and various modifications or changes suggested to personsskilled in the art are to be included within the spirit and purview ofthis application and scope of the appended claims.

What is claimed is:
 1. A method for identifying a subject as a silentcarrier of a target gene null allele comprising: (a) performing aplurality of quantitative nucleic acid amplification reactions, whereineach nucleic acid amplification reaction comprises (i) a genomic DNAsample obtained from a single haploid cell from the subject, (ii) atleast one pair of oligonucleotide primers for amplification of a targetregion of a target gene, wherein the target region is absent in a targetgene null allele, and (iii) at least one pair of oligonucleotide primersfor amplification of a target region of a reference gene; (b) detectingthe presence or absence of the target gene amplification product; (c)detecting the presence or absence of the reference gene amplificationproduct; (d) determining a ratio of detected target gene amplificationproduct to detected reference gene amplification product; and (e)characterizing the subject as a silent carrier of the target gene nullallele if the ratio of target gene amplification products to referencegene amplification products is at or below a threshold level betweenabout 0.5 and about 0.8.
 2. The method of claim 1, wherein the thresholdlevel is about 0.75.
 3. The method of claim 1, wherein the ratio oftarget gene amplification products to reference gene amplificationproducts in a silent carrier of a target gene null allele isapproximately 0.5.
 4. The method of claim 1, wherein the haploid cell isa naturally occurring gamete cell or an induced haploid cell.
 5. Themethod of claim 4, wherein the haploid cell is a sperm cell.
 6. Themethod of claim 4, wherein the induced haploid cell is derived from aninduced pluripotent stem cell (iPSC).
 7. The method of claim 1, whereinthe subject is a human subject.
 8. The method of claim 1, wherein thereference gene is selected from among CFTR, GAPDH, HMBS, B2M, HPRT1,RPL13A, SDHA, TBP, UBC, YWHAZ, PRDX6, ADD1, HLA-A, RAD9A, ARHGEF7,EIF2B2, PSMD7, BCAT2, and ATP5O.
 9. The method of claim 1, wherein thetarget gene is SMN1.
 10. The method of claim 1, wherein the target geneamplification product comprises exon 7 of SMN1 or a portion thereof. 11.The method of claim 1, wherein homozygous deletion of the target gene isassociated with a disease or condition.
 12. The method of claim 11,wherein the disease or condition is spinal muscular atrophy (SMA). 13.The method of claim 1, wherein at least one oligonucleotide primer foramplification of the target gene is labeled with a detectable moiety.14. The method of claim 1, wherein at least one oligonucleotide primerfor amplification of the reference gene is labeled with a detectablemoiety.
 15. The method of claim 13 or claim 14, wherein the detectablemoiety is a fluorescent moiety.
 16. The method of claim 1, wherein thequantitative nucleic acid amplification reaction is quantitative PCR.17. The method of claim 1, wherein each nucleic acid amplificationreaction is performed in a separate well of a multiwell plate.
 18. Themethod of claim 1, wherein each haploid cell is encapsulated in amicrodroplet.
 19. The method of claim 18, wherein the microdroplets aredispersed in an aqueous-in-oil emulsion.
 20. The method of claim 18,wherein the quantitative nucleic acid amplification reaction is dropletdigital PCR.
 21. The method of claim 1, wherein the target geneamplification product is detected with a labeled nucleic acid probespecific for the target gene amplification product.
 22. The method ofclaim 1, wherein the reference gene amplification product is detectedwith a labeled nucleic acid probe specific for the reference geneamplification product.
 23. The method of claim 1, wherein the methodfurther comprises determining the copy number of the target gene in adiploid cell from the subject.
 24. The method of claim 1, wherein themethod further comprises generation of a report, wherein the reportcontains an assessment of the likelihood that the subject is a silentcarrier of the target gene null allele.
 25. The method of claim 1,wherein the method comprises a step of preparing the genomic DNA fromsingle haploid cells.
 26. The method of claim 25, wherein preparing thegenomic DNA from single haploid cells comprises: (a) sorting singlehaploid cells into separate reaction vessels at a concentration of onehaploid cell per reaction vessel; and (b) contacting each sorted cellwith a lysis buffer to release the genomic DNA from the cell.
 27. Themethod of claim 26, wherein the lysis buffer comprises an enzyme. 28.The method of claim 27, wherein the lysis buffer comprises proteinase K.29. The method of claim 26, wherein preparation of the genomic DNA andthe nucleic acid amplification reaction are performed in the samereaction vessel.
 30. The method of claim 26, wherein preparation of thegenomic DNA and the nucleic acid amplification reaction are performed inseparate reaction vessels.
 31. The method of claim 26, wherein thereaction vessel is a microtiter plate.
 32. The method of claim 25,wherein preparing the genomic DNA from single haploid cells comprises:(a) encapsulating single haploid cells into aqueous microdroplets at aconcentration of one haploid cell per reaction vessel; and (b)contacting each single haploid cell within each microdroplet with alysis buffer to release the genomic DNA from the cell.